Project Summary The ability of self-renewal and the capability to differentiate into mature cell types are two critical properties of stem cells. Tissue-derived stem cells (TDSCs) possess multipotent differentiation capability, which is the most crucial property to make them a valuable cell source for treatments of diseases or injuries. Usually, only limited quantities of primary stem cells can be directly isolated from tissues; therefore, in vitro expansion of primary stem cells is needed to obtain large quantities of cells for therapeutic applications. However, in vitro expansion of TDSCs results in the cells to lose their differentiation capability, which makes a generation of large quantities of high-potential stem cells difficult. This dramatically hampers the therapeutic applications of the TDSCs. Our long- term goals are: (a) To elucidate the molecular mechanisms causing the loss of differentiation in TDSCs during expansion. (b) To develop methods to preserve their differentiation capability, such that large quantities of high- potency TDSCs can be obtained from in vitro expansion for cell-based therapies. In preliminary studies, we found that expression of cysteine-rich secretory protein LCCL domain-containing-2 (Crispld2) is dramatically decreased in major TDSCs when the cells lose their osteogenic capability after in vitro expansion. More importantly, knockdown of this gene in high potential osteogenic TDSCs causes the cells to lose their osteogenic differentiation ability. Crispld2 knockout was reported to be embryonic lethal, suggesting its crucial role in development. Our central hypothesis is that in vitro expansion of TDSCs results in dysregulation of Crispld2, which in turn leads to loss of their differentiation ability. The hypothesis will be tested in two specific aims with human bone marrow stem cells (hBMSCs): Aim 1. Identify and characterize transcription factors (TFs) regulating Crispld2 expression in hBMSCs. Aim 2. Determine if enforced or induced expression of Crispld2 affects differentiation of hBMSCs. We will combine novel and modern technologies, CRISPR/Cas9, and bioinformatics, with traditional cell and molecular biology methods, such as cell culture, bioassays, gene expression analysis, and cell differentiation assays to achieve these Aims. In vitro cell experiments and in vivo animal studies will be used in our approaches. This proposal is significant because it will lead to overcoming the difficulty in generating large quantities of TDSCs by understanding the molecular regulation for maintaining the differentiation capability of the stem cells. We expect to identify TFs (activators and repressors) that regulate Crispld2 expression and determine whether high-level Crispld2 expression is necessary to preserve the differentiation capability of TDSCs. Accomplishment of this project will begin to elucidate the regulatory network of Crispld2. Because Crispld2 is widely expressed in many tissues and has multiple functions, including development, cellular defense, stem cell differentiation, cell signal transduction, etc., the results of this project to define the regulatory factors of crsipld2 will have a broad and significant impact and implication. Furthermore, the study will quest the potential in using estrogen and progesterone to preserve the differentiation ability of TDSCs.